Intramolecular Interaction with the E6 Region Stabilizes the Closed Conformation of the N-SH2 Domain and Concurs with the Self-Inhibitory Docking in Downregulating the Activity of the SHP2 Tyrosine Phosphatase: A Molecular Dynamics Study

The localization and activity of the SHP2 tyrosine phosphatase across different cellular compartments to the target substrates are steered by the binding of phosphotyrosine (pY) peptides to the tandem SH2 domains. The most N-terminal domain (N-SH2) can also keep the enzyme inactive by intramolecular occlusion of the catalytic site. Enzyme activity can be recovered by an allosteric disruption of this self-inhibitory docking upon the binding of pY peptides to the N-SH2 domain. Prior to this, the N-SH2 domain must abandon the closed conformation because it impedes the access of pY peptides to the binding cleft. Although it cooperates with the self-inhibitory docking in the negative regulation of the phosphatase activity, the structural determinants of the stability of the closed conformation in the self-inhibited phosphatase are still elusive. To address this issue, a molecular dynamics simulation study is carried out. It is shown that the closed conformation is stabilized by the interaction of the N-SH2 domain with a conserved peptide portion in the region encoded by PTPN11 exon 6 (E6).


Introduction
Protein tyrosine phosphorylation modulates many protein features, such as stability, subcellular localization, patterns of molecular recognition, activity, and other functions contributing to signaling and biological regulation [1]. In concert with tyrosine kinases, which add a phosphate group, the protein phosphorylation status is controlled by protein tyrosine phosphatases, which catalyze the removal of phosphate groups via a cysteinylphosphate intermediate [2]. The Src homology 2 (SH2) domain containing phosphatase 2 (SHP2, also known as tyrosine-protein phosphatase non-receptor type 11) is encoded by the PTPN11 gene and characterized by two SH2 domains arranged in tandem (N-SH2, a.a. 1-103, and C-SH2, a.a. 111-213) N-terminally to the phosphatase domain (PTP, a.a. 253-529). SHP2 has functions ranging across the regulation of cell growth, tissue inflammation, cell survival in oxidative stress pathways [3], hematopoiesis and differentiation [4,5], cell proliferation [6], and migration [7].
Mutations in the PTPN11 gene causing a loss of phosphatase activity are associated with LEOPARD syndrome [8], while those enhancing the activity are linked to Noonan syndrome [9]. Furthermore, mutations incrementing the phosphatase activity are common in juvenile myelomonocytic leukemia [10], and this led to classifying PTPN11 as an oncogene. Mutations enhancing the enzyme activity are also found in some human solid tumors (e.g., lung cancer, colon cancer, neuroblastoma, and melanoma) and adult acute myelogenous leukemia [11]. The oncogenic role of PTPN11 was also supported by the finding that leukemia-associated mutations prolong cell survival [12] and cause defects in since substrate dephosphorylation still occurs even without most E6 residues, as shown by the SHP2 constructs formed by residues 230-593 [36] and residues 246-547 [37]. E6 conservation among tyrosine phosphatases featuring tandem SH2 domains (SHP1, SHP2, and corkscrew) would suggest importance for this region, but its function remains elusive. To examine whether E6 has a role in the closed conformation, molecular dynamics (MD) simulations were carried out on the N-SH2 domain in different structural contexts: isolated N-SH2 (N-SH2closed, closed conformation; N-SH2open, open conformation), wild-type self-inhibited SHP2 (SHP2-wt), self-inhibited SHP2 carrying mutations weakening the interaction of E6 with N-SH2 (SHP2-W248A and SHP2-W248A/E252A), and self-inhibited SHP2 fully deprived of this interaction (E6 deletion construct, SHP2-ΔE6). bound to a pY peptide (PDB 1AYA). The domains are in distinct colors; E6 is in red, and inter-domain linkers are in white. The close-up view highlights E6 and the invariant 246-GFWEEFE-255 segment in more detail. (bottom) Sequence alignment of SHP2, SHP1, and corkscrew with secondary structure elements and amino acid conservation (displayed on top of residues), with ranges of domains and inter-domain linkers (colored bars below the alignment). Mutated and disordered residues in the SHP2 crystal structure are highlighted in red and gray, respectively. The 139 amino acid-long insertion in the corkscrew sequence is omitted (its position is indicated).

2.2.
Interactions of E6 with N-SH2, C-SH2, and PTP in the Wild-Type Self-Inhibited SHP2 In the self-inhibited enzyme, E6 interacts with all three domains of the SHP2 phosphatase ( Figure 1). The residues involved in the interactions of E6 with each domain in the SHP2 crystal structure and at various times of the MD simulation of SHP2-wt are shown in Figure 2 (the individual names of the interacting residues are indicated in correspondence with each heat map row). The most extended interface of interaction with E6 involved the PTP domain, while only a few E6 residues interacted with either N-SH2 or C-SH2. In particular, during the simulation, only one E6 residue at the E6/N-SH2 interface, Trp248, presented stable interaction with N-SH2, while other residues interacted transiently (Lys235, Gln245, Phe251, and Glu252). In the N-SH2 domain, most of the residues interacting with E6 were located in the loop formed by residues 34-40.

Domain-Domain Interactions in the Self-Inhibited SHP2: Comparison of Wild-Type and E6 Deletion Constructs
To determine the importance of the E6 region in the various domain-domain interactions in the self-inhibited enzyme, the MD simulations of SHP2-wt and SHP2-∆E6 were compared to identify possible differences in the inter-domain interfaces.

Interactions between the N-SH2 and C-SH2 Domains
The N-SH2 and C-SH2 domains are held together by a short linker and few noncovalent interactions (dominated by the salt bridge between Arg5 and Asp192). No major changes in the N-SH2/C-SH2 interface were observed in the MD simulations of SHP2-wt nor in the SHP2-∆E6 construct ( Figure S2).

Interactions between the C-SH2 and PTP Domains
The C-SH2/PTP interface did not change appreciably in the MD simulations of SHP2wt but increased in extension in the simulation of SHP2-∆E6 ( Figure S3). This is an obvious consequence of the deletion of E6, which in the wild-type protein lays between C-SH2 and PTP and physically separates the two domains. This suggests that one role of E6 could be to prevent C-SH2 from collapsing on the PTP domain, which might abnormally hinder functional regions.

Interactions between the N-SH2 and PTP Domains
The largest inter-domain interface in the self-inhibited SHP2 is the one formed by N-SH2 and PTP. Despite E6 appearing to collate the two domains, thus stabilizing the inactive enzyme configuration, the N-SH2/PTP interface did not differ appreciably in the SHP2-wt or SHP2-∆E6 constructs ( Figure S4), nor were there significant changes in the N-SH2/PTP area of contact ( Figure S5) within the time length of these simulations.  Interacting residues are listed next to each heat map. At the side of each heat map are shown the molecular surfaces of E6 or the domains isolated from the SHP2 crystal structure and from the MD snapshots of SHP2-wt at 3 ns (ca. the time to achieve equilibration as assessed from RMSDs) and at 30 ns. These surfaces are colored according to the shortest distance from the interacting partner (E6 or a domain), employing the same color key as for the heat maps. The surfaces with mapped distances are rotated (as indicated by arrows) with respect to the parent multi-domain structure at the top of each figure section to obtain frontal view of relevant interacting residues.

Exploration of Constructs Presenting N-SH2 in a Different Structural Context
The synchronism with the self-inhibitory docking suggests that the closed conforma-  Table 1).

RMSDs of the N-SH2 Domain
The RMSDs of the N-SH2 domain in the self-inhibited constructs (SHP2-wt, SHP2-∆E6, SHP2-W248A, and SHP2-W248A/E252A) and in the isolated N-SH2 domain (N-SH2 closed and N-SH2 open ) did not differ significantly if calculated using the structures kickstarting the respective MD simulations as references ( Figure S1). However, by recalculating the RMSDs of N-SH2 of SHP2-wt and SHP2-∆E6 relative to the crystal structure of N-SH2 in open conformation (PDB 1AYA), an important decrement in the atomic displacements was seen in the E6 deletion construct but not in the wild-type SHP2 construct, and the source of this decrease could be identified in the loops formed by residues 34-40 and residues 65-69 ( Figure S6). The change in the RMSDs of the 34-40 loop is consequential to the interaction of the N-SH2 region with E6, which can occur in the self-inhibited SHP2 but not in the E6 deletion construct. Quite surprisingly, the RMSD change was localized in amino acids 65-69 despite they did not interact with E6. The latter region is functionally important as it encompasses the EF loop functional in the closed or open conformation of the N-SH2 domain. When comparing SHP2-wt and SHP2-∆E6, no difference could be seen in the RMSDs of the other two regions deemed important for N-SH2 functions, the BG loop (also determining the closed or open conformation), and the D'E loop (inhibiting the phosphatase catalytic pocket). The decreased RMSDs in SHP2-∆E6 could suggest that this construct gained structural similarity with the crystal structure of the open conformation N-SH2 domain.

Volumes of the Groove in the pY Peptide-Binding Region of the N-SH2 Domain
To assess whether the deletion of E6 might indeed favor the change from closed to open conformation in the self-inhibited enzyme, the volume of the groove in the pY peptide-binding region of the N-SH2 domain was calculated and averaged from MD simulation snapshots. An increase in the volume could be observed in SHP2-∆E6 compared with the three distinct simulations of SHP2-wt ( Figure 3), indicating that the E6 deletion construct enlarged the space available between the BG and EF loops. It must be underscored that the modeled construct carrying such an important deletion was not intended to reproduce what would be the fold of the real protein construct. The SHP2-∆E6 model was rather designed to examine how the N-SH2 domain would behave after the "sudden" disappearance of its interactions with E6 starting from the multi-domain protein prearranged in its native, self-inhibited configuration. As a matter of fact, incremented volumes were also observed in the self-inhibited structures carrying the much simpler single-and double-missense mutations W248A and W248A/E252A. As can be seen in the heat maps of Figure 2, Trp248 and Glu252 were E6 residues both contributing to the interactions with the N-SH2 domain such that their replacement with an alanine likely weakened such interactions. Comparing the SHP2-W248A and SHP2-W248A/E252A mutants, the latter had the most increased volume. This suggests that the two missense mutations, by cumulatively weakening the E6/N-SH2 interaction, can synergistically induce enlargement of the space used for pY peptide binding, which became similar to the open conformation structure N-SH2 open . The latter maintained its large initial volume, which is characteristic of the open conformation. Coherence with the conformation of their parent structures was also exhibited by SHP2-wt and N-SH2 closed , both preserving the small volume typical of the closed conformation. The statistical significance of an outcome in which all three simulations of the wild-type self-inhibited structure maintained the closed conformation and the simulations of all three mutated self-inhibited structures exhibited closed-to-open conversion was 0.05 according to the hypergeometric test. Taken together, these results indicate that the interaction of E6 with the N-SH2 domain has a role in the stability of the closed conformation in the self-inhibited SHP2 tyrosine phosphatase.    Guvench et al. [38] discovered a key role of Tyr66 in the regulation of the EF loop functional conformations, determining that the open conformation is settled when the tyrosyl group is pulled away from the pY peptide-binding cleft. This is achieved by a set of side chain-side chain non-bonding interactions involving Tyr66: hydrogen bonding with Asp40, either hydrogen bonding or hydrophobic interaction with Lys55, and either hydrogen bonding or pi stacking with Gln57 ( Figure 4). Conversely, mutual interactions among Asp40, Lys55, and Gln57 set free the tyrosyl group allowing the N-SH2 domain to regain the closed conformation. It is still unknown what drives Asp40, Lys55, and Gln57 rearrangement in favor of their mutually interacting configuration. It would be valuable to know the mechanism given the importance of the closed conformation. As a matter of facts, this conformation disfavors the recruitment of pY ligands by N-SH2 and delays undocking of this domain from PTP, thus cooperating with self-inhibition to negatively regulate the enzyme activity. It is also puzzling that the open and closed conformations are both thermodynamically stable, as evidenced by crystallographic and MD simulation studies. It would rather be expected that the enzyme could rapidly swap between the active and inactive states. This would ensure responsiveness to sudden changes in pY ligand types and concentrations in the diverse cellular microenvironments and physiological conditions encountered by the enzyme. As a matter of fact, for such a pleiotropic enzyme, it is necessary to process substrates as well as to avoid indiscriminate dephosphorylations in a timely manner. After all, abnormally high or low phosphatase activity both characterize a number of disease-associated PTPN11 mutations.  Figure 6). Taking into account the different N-SH2 structural contexts examined in this study (Table 1), these results highlight that the closed conformation, stable in the self-inhibited enzyme, could switch with ease to the open conformation upon weakening or abolishing the interactions of E6 with the N-SH2 domain. rearrangement in favor of their mutually interacting configuration. It would be valuable to know the mechanism given the importance of the closed conformation. As a matter of facts, this conformation disfavors the recruitment of pY ligands by N-SH2 and delays undocking of this domain from PTP, thus cooperating with self-inhibition to negatively regulate the enzyme activity. It is also puzzling that the open and closed conformations are both thermodynamically stable, as evidenced by crystallographic and MD simulation studies. It would rather be expected that the enzyme could rapidly swap between the active and inactive states. This would ensure responsiveness to sudden changes in pY ligand types and concentrations in the diverse cellular microenvironments and physiological conditions encountered by the enzyme. As a matter of fact, for such a pleiotropic enzyme, it is necessary to process substrates as well as to avoid indiscriminate dephosphorylations in a timely manner. After all, abnormally high or low phosphatase activity both characterize a number of disease-associated PTPN11 mutations.    are shown. The fitting was made by introducing a set of Gaussians (an identical number of Gaussians within a column of plots) and seeking squared minimization (globally for all plots in a column). In this procedure, the amplitude was allowed to vary independently for all Gaussians in all plots in a column, and their widths and centers were also varied independently within single plots but synchronizing these changes in the corresponding Gaussians of all other plots in the same column. With these constraints, the populations of the conformational ensembles characterized by particular loop/loop or residue/loop separations could be compared among the various MD simulations through the amplitudes of corresponding Gaussians in the same column of plots. and their sums (red line) are shown. The fitting was made by introducing a set of Gaussians (an identical number of Gaussians within a column of plots) and seeking squared minimization (globally for all plots in a column). In this procedure, the amplitude was allowed to vary independently for all Gaussians in all plots in a column, and their widths and centers were also varied independently within single plots but synchronizing these changes in the corresponding Gaussians of all other plots in the same column. With these constraints, the populations of the conformational ensembles characterized by particular loop/loop or residue/loop separations could be compared among the various MD simulations through the amplitudes of corresponding Gaussians in the same column of plots. Figure 6. Averaged MD conformers of the N-SH2 domain from the simulations of the self-inhibited SHP2 and its mutants with weakened or annulled interactions between E6 and N-SH2 (averages were made on the conformers, collected every 5000 femtoseconds in the 3-30-ns time interval and previously aligned across N-SH2 domain residues 3-103 to the structures kickstarting the respective MD simulations). A larger ribbon thickness indicates a higher RMSD (calculated at C α atoms). For Figure 6. Averaged MD conformers of the N-SH2 domain from the simulations of the self-inhibited SHP2 and its mutants with weakened or annulled interactions between E6 and N-SH2 (averages were made on the conformers, collected every 5000 femtoseconds in the 3-30-ns time interval and previously aligned across N-SH2 domain residues 3-103 to the structures kickstarting the respective MD simulations). A larger ribbon thickness indicates a higher RMSD (calculated at C α atoms). For the sake of clarity, pairwise structural superpositions between the wild-type SHP2-wt protein and each mutant (SHP2-W248A, SHP2-W248A/E252A, and SHP2-∆E6) are presented. The distance between the EF and BG loops (measured between the C α atoms of Gly67 and Asn92) is labeled on each averaged structure.

The Role of the Cation-pi Interaction between Lys55 and Tyr66
A finding emerging from this study is that the N-SH2 closed conformation was stabilized by interactions with E6. Since the EF loop was relatively distant from the N-SH2/E6 interface, this effect should be mediated allosterically. Guvench et al. [38] determined that the critical EF loop residue Tyr66 acts as a conformational switch, which can select the open conformation by undergoing side chain-side chain interactions with Asp40, Lys55, and Gln57. In the present MD study, two additional and distinct interactions also involving Tyr66 and Lys55 were found. One was hydrogen bonding of Lys55 oxygen with Tyr66 nitrogen, which occurred in all closed conformation constructs but not in the open conformation N-SH2 (see Lys55-Tyr66 distance plot in Figure 5) and thus appearing to be in competition with the above-mentioned interactions stabilizing the open conformation. The second was the Lys55/Tyr66 cation-pi interaction, which was observed with a comparable frequency in N-SH2 closed , SHP2-wt, SHP2-W248A, and SHP2-W248A/E252A, less frequently in SHP2-∆E6, and more rarely in N-SH2 open (Figure 7). Since the cation-pi interaction can exert attractive forces at longer distances than the other non-bonding interactions mutually attracting Lys55 and Tyr66, it might serve to enhance the ability of the two residues to join together after they are separated by the domain's conformational changes. In particular, the Lys55/Tyr66 cation-pi interaction can aid the inter-conversion between closed and open conformations in both directions. In fact, in the closed conformation, the Tyr66 and Lys55 side chains were quite distant (Figure 4), and thus the cation-pi interaction was the first to mediate the mutual attraction of the two residues. Thereafter, the tyrosyl group could eventually shift to the alternative short-range interactions, with Lys55 also engaging Asp40, and Gln57 for full stabilization of the open conformation. On the other hand, after unloading the pY peptides, the N-SH2 domain might linger in the open conformation, owing to the stabilizing short-range interactions of Tyr66 with Asp40, Lys55, and Gln57. In this circumstance, the Lys55/Tyr66 cation-pi interaction can competitively step in and drive the domain into the closed conformation. Thus, by exchanging the type of nonbonding intra-domain interactions with Tyr66, other critical N-SH2 residues might support smooth conversions between the two functional conformations of the domain. To remark on the importance of this cation-pi interaction, cationic and aromatic residues at positions homologous to Lys55/Tyr66 are also present in the other phosphatases characterized by tandem SH2 domains, such as SHP1 and corkscrew.

Discussion
Guvench et al. [38] reported that Tyr66 determines the closed conformation, owi to intra-domain interactions of the tyrosyl group with the side chains of the Asp40, Lys and Gln57 triad (Figure 4). Given the importance of Tyr66 as a closed or open conform tional switch, it is necessary to understand how the configuration of this tyrosine cou be influenced by the somehow distant interactions between E6 and N-SH2. The triad

Discussion
Guvench et al. [38] reported that Tyr66 determines the closed conformation, owing to intra-domain interactions of the tyrosyl group with the side chains of the Asp40, Lys55, and Gln57 triad (Figure 4). Given the importance of Tyr66 as a closed or open conformational switch, it is necessary to understand how the configuration of this tyrosine could be influenced by the somehow distant interactions between E6 and N-SH2. The triad of residues interacting with Tyr66 can undergo mutual interactions, and one of them, Asp40, is part of the 34-40 loop. Thus, the configuration of the Asp40, Lys55, and Gln57 triad has a direct dependance on the conformation of the 34-40 loop, which can change upon interaction with the extra-domain regions of the phosphatase. Insights were obtained by analyzing how this loop moves relative to the critical residues in the MD simulations of the constructs presenting the N-SH2 domain in different structural contexts. In the cases of SHP2-wt and N-SH2 open , the distance of the 34-40 loop from the D'E loop was similar to that observed in the respective parent crystal structures ( Figure 5). In SHP2-wt, this was due to the restraints imposed on the two loops by their contact with E6 and PTP. In this case, the 34-40 loop protruded outward from the N-SH2 domain surface to gain favorable interactions with E6. However, when the interaction with E6 was weakened or annulled, as in SHP2-W248A, SHP2-W248A/E252A, and SHP2-∆E6, the 34-40 loop bent inward, achieving a stabilizing intra-domain contact with Lys55. This was evidenced by the decreased distance between the C α atoms of Ser36 (residue in the 34-40 loop) and Lys55, which was observed in all three mutants ( Figure 5). The bending of the 34-40 loop caused the relocation of Asp40 and the twisting of the β-strand containing Lys55 and Gln57. As a result, Asp40, Lys55 and Gln57 were brought closer to the Tyr66 tyrosyl group. Thus, weakening or annulling the interaction of E6 with N-SH2 in the self-inhibited enzyme had the final effect to bring the three residues into the configuration that stabilized the open conformation that was described by Guvench et al. [38]. This particular configuration was stable in the MD simulation of SH2 open . Weakening or abrogating E6/N-SH2 interactions also caused the 34-40 loop to relax onto the D'E loop, as observed in SHP2-W248A, SHP2-W248A/E252A, and SHP2-∆E6 (see the decreased distances between the C α atoms of Pro38 and Thr59 in Figure 5). With respect to the separation of the D'E and 34-40 loops, a striking difference could be observed in the simulations of the two isolated N-SH2 domain constructs. In N-SH2 open the 34-40 loop maintained a constant distance from the D'E loop, whereas in N-SH2 closed the two loops became much closer. SH2 closed was also the only structure that lost the hydrogen bond between the Thr59 nitrogen and Tyr62 backbone oxygen (highlighted by the sudden increase in the Thr59-Tyr62 distance in Figure 5). This conferred the D'E loop with a peculiar conformation among all constructs. The unusual behavior of the D'E loop in N-SH2 closed might be an artifact arising from the atypical structural context of this domain construct. In fact, N-SH2 closed was obtained by computational truncation of the self-inhibited SHP2-wt, which produced an isolated N-SH2 domain yet was conformationally frozen as if it was still bound to PTP and E6. Instead, in the undocking path of the real enzyme, it can be thought that while the N-SH2 domain gradually reduces its contact with the extra-domain regions PTP and E6, its conformation likely undergoes stepwise adaptations. Thus, when interpreting the MD simulation of N-SH2 closed , it must be taken into account that the construct does not represent an N-SH2 domain in full native conditions. It can be deduced that the stability of the closed conformation in the self-inhibited enzyme is not an intrinsic property of the N-SH2 domain but the consequence of its intramolecular interactions with the extra domain region E6. In particular, Tyr66 conformation is allosterically modulated by the interaction of E6 with the 34-40 loop.
The E6 deletion construct (SHP2-∆E6) was obtained by removing residues 215-252 from SHP2-wt, followed by conjugation of the deletion boundaries Gln214 and Thr253. Since these two residues were proximal in the crystal structure, their conjugation required applying geometry optimization to them plus a few flanking residues (a.a. 212-214 and 253-255) and to the side chain of Arg111 that lays between Gln214 and Thr253 ( Figure S7). The SHP2-W248A and SHP2-W248A/E252A mutants were obtained from SHP2-wt by introducing the single-and double-missense mutations W248A and W248A/E252A. The The molecular structures were embedded in explicit water solvent (TIP3P water model) with Na + and Cl − ions added to achieve electroneutrality and an ionic strength of 0.1 mol/L. Before the MD simulations, each molecular system was subjected to two cycles of minimization, equilibration, and minimization. In the first cycle, all protein atoms were restrained, while the water and ions were allowed to move freely. In the second cycle, only the protein backbone atoms were restrained (in these cycles, minimizations and equilibrations were carried out for 20,000 and 500,000 steps, respectively). Finally, all restraints were removed, and the systems were minimized again for 20,000 steps. The MD simulations were performed with NAMD (v.2.8) [40,41], employing the CHARMM22 protein force field [42] including CMAP correction under periodic boundary conditions using an integration step of 1 femtosecond and flexible bonds at a temperature T = 310 K, controlled with a Langevin thermostat, and using a damping coefficient of 1 ps −1 . Short-range interactions were computed at every time step, and long-range electrostatic interactions were computer every two time steps, employing a switching distance of 10 Angstroms, cutoff of 12 Angstroms, and pair list distance of 13.5 Angstroms. The atomic coordinates were recorded every 5000 femtoseconds.

Comparison of the Domain-Domain and E6-Domain Interfaces in the MD Simulations of SHP2-wt and SHP2-∆E6
The domain-domain and E6-domain interfaces in the self-inhibited SHP2 crystal structure and in the MD snapshots of SHP2-wt and SHP2-∆E6 were identified by determining all residues in a region (domain or E6) falling within a 5-Å threshold from a distinct region. For each region, the distances of the interfacial residues from the closest heavy atom in the interacting region partner were recorded and represented in the figures by heat maps and molecular surfaces with distance-based colors. The linkers between N-SH2 and C-SH2 (a.a. 104-110), between C-SH2 and E6 in SHP2-wt (Gln 214), and between C-SH2 and PTP in SHP2-∆E6 (Thr253) were not included in the interfacial residues.

N-SH2/PTP Area of Contact in the MD Simulations of SHP2-wt and SHP2-∆E6
The interfacial area between the N-SH2 and PTP domains was calculated as follows: where A(i) N-SH2 , A(i) PTP , and A(i) N-SH2/PTP represent, respectively, the van der Waals surface areas of the isolated N-SH2 domain, isolated PTP domain, and the isolated intramolecular N-SH2/PTP complex at simulation time i.

Volume of the N-SH2 pY Peptide-Binding Cleft in the MD Simulations
The pY peptide-binding cleft in the N-SH2 domain was identified in the groove lining between the BG and EF loops. The volume of this groove was calculated with Swiss-PdbViewer [43] on the MD conformers of N-SH2 closed , SHP2-wt (triplicate MD runs), SHP2-W248A, SHP2-W248A/E252A, SHP2-∆E6, and N-SH2 open recorded every 1 ns from 3 ns (ca. the time to achieve equilibration) to 30 ns.

Identification of the MD Conformers Showing Lys55/Tyr66 Cation-Pi Interaction
The MD conformers presenting a cation-pi interaction between Lys55 and Tyr66 were identified based on the geometric conditions L ≤ 8 Å and θ ≤ 60 • (Figure 7), where L represents the distance between the positive electric charge (lysine N ζ atom) and the π cloud center (tyrosine aromatic ring center) and θ is the angle between the normal to the plane of the aromatic ring and the segment connecting the positive charge with the center of the aromatic ring. The fractions of the conformers presenting the Lys55/Tyr66 cation-pi interaction were calculated over the MD snapshots (recorded every 5000 femtoseconds) in the 3-30-ns time interval of the simulations.

Conclusions
This study unravels an allosteric mechanism for the stabilization of the closed con- It is important to know the structural determinants of the closed conformation since it delays the binding of pY peptides on the N-SH2 domain, thus cooperating with selfinhibition in the negative regulation of the enzyme. E6 conservation in phosphatases sharing the same tandem SH2 domain architecture suggests that this module is adapted to tune N-SH2 conformation to confer these enzymes with enhanced versatility of regulation and transport across cellular compartments.
Further studies of the complex functions of this enzyme, in particular to gather useful information on the conformational sampling of domains and their interactions with ligands, may take advantage of MD simulations followed by free energy analysis [44,45].